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Isoflavones such as genistein and daidzein prevented agonist-induced vascular contraction in isolated rat aortic rings. Glyceollins are derived from the parent isoflavone daidzein through a series of pterocarpan intermediates. We hypothesized that glyceollin attenuates vascular contractions through inhibition of RhoA/Rho kinase pathway. Rat aortic rings were denuded of endothelium, mounted in organ baths and treated with either glyceollin (20 or 100 μM) or vehicle (DMSO) for 60 min after submaximal contraction by NaF (8.0 mM). The phosphorylation level of the myosin light chain (MLC20), myosin phosphatase target subunit 1 (MYPT1) and protein kinase C (PKC)-potentiated inhibitory protein for heterotrimeric myosin light chain phosphatase of 17-kDa (CPI17) were determined by means of the Western blot. Glyceollin not only inhibited vascular contractions induced by NaF (8.0 mM), but also decreased the activation of RhoA and subsequent phosphorylation of MYPT1Thr855 and CPI17Thr38. These results indicate that glyceollin attenuates vascular contraction by inhibiting RhoA/Rho-kinase signaling.